The enteric pathogen Shigella is one of the leading causes of moderate-to-severe diarrhea and death in young children in developing countries. Transformed cell lines and animal models have been widely used to study Shigella pathogenesis. In addition to altered physiology, transformed cell lines are composed of a single cell type that does not sufficiently represent the complex multicellular environment of the human colon. Most available animal models do not accurately mimic human disease. The human intestinal enteroid model, derived from LGR5+ stem cell-containing intestinal crypts from healthy subjects, represents a technological leap in human gastrointestinal system modeling and provides a more physiologically relevant system that includes multiple cell types and features of the human intestine. We established the utility of this model for studying basic aspects of Shigella pathogenesis and host responses. In this study, we show that Shigella flexneri is capable of infecting and replicating intracellularly in human enteroids derived from different segments of the intestine. Apical invasion by S. flexneri is very limited but increases ∼10-fold when enteroids are differentiated to include M cells. Invasion via the basolateral surface was at least 2-log10 units more efficient than apical infection. Increased secretion of interleukin-8 and higher expression levels of the mucin glycoprotein Muc2 were observed in the enteroids following S. flexneri infection. The human enteroid model promises to bridge some of the gaps between traditional cell culture, animal models, and human infection.

The objective of this study was to determine if a quantitative scoring system for evaluation of hemosiderin content of alveolar macrophages obtained by bronchoalevolar lavage provides a more sensitive test for the detection of exercise-induced pulmonary hemorrhage (EIPH) in horses than does endoscopy of the lower airways. A sample population composed of 74 Standardbred racehorses aged 2-5 years was used. Horses were grouped as either control (EIPH-negative) or EIPH-positive based on history and repeated postexertional endoscopic evaluation of the bronchial airways. Bronchoalveolar lavage was performed and cytocentrifuge slides were stained with Perl's Prussian blue. Alveolar macrophages were scored for hemosiderin content by a method described by Golde and associates to obtain the total hemosiderin score (THS). Test performance criteria were determined with a contingency table. All subjects had some degree of hemosiderin in the alveolar macrophages, regardless of group. The distribution of cells among the different grades followed a significantly different pattern for the control group versus horses with EIPH (P Ͻ .05). When using a THS of 75 as a cutoff point, the THS test was found to have a sensitivity of 94% and a specificity of 88%. The level of agreement beyond chance, between the EIPH status and the THS test result was very good (Cohen's kappa ϭ 74%). The conclusion was made that careful assessment and scoring of alveolar macrophages for hemosiderin by means of the Golde scoring system shows promise as a more sensitive approach than repeated postexertional endoscopy alone to detect EIPH.

Background:Bone-metastatic, castration-resistant prostate cancer (bmCRPC) represents a lethal stage of the most common noncutaneous cancer in men. The recent introduction of Radium-223 dichloride, a bone-seeking alpha particle (α)–emitting radiopharmaceutical, demonstrates statistically significant survival benefit and palliative effect for bmCRPC patients. Clinical results have established safety and efficacy, yet questions remain regarding pharmacodynamics and dosing for optimized patient benefit.Methods:We elucidated the biodistribution of 223Ra as well as interaction with the bone and tumor compartments in skeletally mature mice (C57Bl/6 and CD-1, n = 3–6) and metastasis models (LNCaP and PC3, n = 4). Differences in uptake were evaluated by µCT and histological investigation. Novel techniques were leveraged on whole-mount undecalcified cryosections to determine microdistribution of Radium-223. All statistical tests were two-sided.Results: 223Ra uptake in the bones (>30% injected activity per gram) at 24 hours was also accompanied by non-negligible remnant activity in the kidney (2.33% ± 0.36%), intestines (5.73% ± 2.04%), and spleen (10.5% ± 5.9%) Skeletal accumulation across strains did not correspond with bone volume or surface area but instead to local blood vessel density (P = .04). Microdistribution analysis by autoradiography and α camera revealed targeting of the ossifying surfaces adjacent to the epiphyseal growth plate. In models of PCa metastasis, radioactivity does not localize directly within tumors but instead at the apposite bone surface. Osteoblastic and lytic lesions display similar intensity, which is comparable with uptake at sites of normal bone remodeling.Conclusions:Profiling the macro- and microdistribution of 223Ra in healthy and diseased models has important implications to guide precision application of this emerging α-therapy approach for bmCRPC and other bone metastastic diseases.

Background: Teaching methods that provide an opportunity for individual engagement and focussed feedback are required to create an active learning environment for case-based teaching in large groups. Aims: A prospective observational controlled study was conducted to evaluate whether the use of an audience response system (ARS) would promote an active learning environment during case-based discussions in large groups, have an impact on student motivation and improve long-term retention. Methods: Group A (N ¼ 83) participated in large group case discussions where student participation was voluntary, while for group B (N ¼ 86) an ARS was used. Data collection methods included student and teacher surveys, student focus group interviews, independent observations and 1-year post-course testing. Results: Results indicated that the use of an ARS provided an active learning environment during case-based discussions in large groups by favouring engagement, observation and critical reflection and by increasing student and teacher motivation. Although final exam results were significantly improved in group B, long-term retention was not significantly different between groups. Conclusions: It was concluded that ARS use significantly improved the learning experience associated with case-based discussions in a large group of undergraduate students.

Results suggested that overall clinical efficacy of a paste formulation of firocoxib in horses with naturally occurring osteoarthritis was comparable to efficacy of a paste formulation of phenylbutazone.

This study was performed to estimate the prevalence of gastric ulcers in Standardbred racehorses, to describe the lesion score and location, and to identify potential risk factors. Two hundred seventy-five (275) Standardbred horses from 5 training centers and 2 racetracks in Quebec, Canada, were studied. Historical data for the 2 months before examination were recorded for each horse, and the presence of gastric ulcers was determined by gastroscopy. A previously reported scoring system that used grades 0-3 for gastric lesions was used. Overall, 121 horses (44.0%; 95% CI, 38.1-50.1%) had gastric ulcers. The prevalence of gastric ulcers was significantly higher (P < .0001) in actively racing horses (63.3%; 95% CI, 54.7-71.2%) than in horses at rest. Multivariate analysis defined that horses in racing (OR = 9.29; 95% CI, 3.55-24.3) were significantly more likely to have gastric ulcers than horses at rest and that trotters (OR = 2.23; 95% CI, 1.28-3.86) were more likely to have gastric ulcers than pacers. The number of lesion sites (P < .0001) and poor body condition (P < .0001) were significantly associated with lesion scores. Gastric ulcers are highly prevalent in Standardbred racehorses. Furthermore, actively racing horses and trotters are more likely to have gastric ulcers. Also, poor body condition in Standardbred racehorses may be an indication that gastric ulcers are present and that lesion scores are high. The cause-and-effect relationship between poor body condition and the presence of gastric ulcers is unclear.

Interactions between the CXCR4 chemokine receptor in breast cancer cells and the ligand CXCL12/SDF-1alpha are thought to play an important role in breast cancer metastases. In this pilot study, CXCR4 expression along with other biomarkers including HER2-neu and EGFR, were measured in primary tumor samples of patients with operable breast cancer to test whether any of these biomarkers alone and in combination could indicate breast cancer with high likelihood of metastasizing to bone marrow. Cytokeratin (CK) positive cells in bone marrow were identified by flow-cytometry following enrichment with CK 7/8 antibody-coupled magnetic beads. Primary tumors (n = 18) were stained with specific antibodies for CXCR4, HER2-neu, EGFR, and PCNA using an indirect avidin-biotin horseradish peroxidase method. The majority of the patients had T2/T3 tumors (72%), or lymph node involvement (67%) as pathologic characteristics that were more indicative of high-risk breast cancer. High CXCR4 cytoplasmic expression was found in 7 of 18 patients (39%), whereas 6 of 18 patients (33%) were found to have CK positivity in bone marrow. The median number of CK(+) cells was 236 (range, 20-847) per 5 x 10(4) enriched BM cells. The presence of CK(+) cells in bone marrow was found to be associated with increased expression of CXCR4 alone or in addition to EGFR and/or HER2-neu expression (P = 0.013, P = 0.005, and P = 0.025, respectively) in primary tumors. Furthermore, three patients with high CK positivity (>236 CK(+) per 5 x 10(4) enriched bone marrow cells) in bone marrow exclusively expressed high levels of CXCR4 with EGFR/HER2-neu (P = 0.001). Our data suggest that high CXCR4 expression in breast cancer may be a potential marker in predicting isolated tumor cells in bone marrow. CXCR4 coexpression with EGFR/HER2-neu might further predict a particular subset of patients with high CK positivity in bone marrow.

Brain metastasis is the most commonly occurring intracranial tumor whose incidence seems to be increasing. With standard therapy, the average survival time of patients is f8 months, and treatment often leads to neurologic dysfunction in longterm survivors, emphasizing the need for novel therapeutics. Clostridium perfringens enterotoxin (CPE) has recently been shown to rapidly and specifically destroy cancer cells expressing CPE receptors claudin-3 and claudin-4. Unfortunately, the utility of CPE is precluded by systemic toxicity because its receptors are expressed in numerous organs. Here, we provide the first preclinical evidence that CPE may be uniquely suited to the local treatment of brain metastasis. By immunohistochemical analysis, claudin-3 and claudin-4 were expressed frequently in metastases from breast (15 of 18), lung (15 of 20), and colon (12 of 14) carcinoma, and infrequently in metastases from renal cell carcinoma (2 of 16) and melanoma (2 of 16). In contrast, expression of claudin-3 and claudin-4 was absent in adjacent normal brain tissue. Further examination of the central nervous system (CNS) revealed low or undetectable levels of claudin-3 and claudin-4 in all regions tested by Western and immunohistochemical analysis. Treatment of breast cancer cell lines (MCF-7, MDA-MB-468, NT2.5-luc) and normal human astrocytes with CPE in vitro resulted in rapid and dose-dependent cytolysis exclusively in breast cancer cells, correlating with claudin-3 and claudin-4 expression. Moreover, intracranial CPE treatment significantly inhibited tumor growth and increased survival in two murine models of breast cancer brain metastasis, without any apparent local or systemic toxicity. These data suggest that CPE therapy may have efficacy against a wide variety of brain metastases without CNS toxicity. [Cancer Res 2007;67(17):7977-82]